Polypeptide complex comprising non-peptidyl polymer having three functional ends

ABSTRACT

Disclosed is a protein complex, comprising a physiologically active polypeptide, a dimeric protein and a non-peptidyl polymer having three functional ends (3-arm), with the linkage of both the physiologically active polypeptide and the dimeric protein to the 3-arm non-peptidyl polymer via respective covalent bonds. The protein complex guarantees the long acting activity and biostability of a physiologically active polypeptide. Having the ability to maintain the bioactivity of physiologically active polypeptides or peptides highly and to significantly improve the serum half life of the polypeptides or peptides, the protein complex can be applied to the development of sustained release formulations of various physiologically active polypeptide drugs. Also, it utilizes raw materials including the physiologically active polypeptides without significant loss, thereby increasing the production yield. Further, it can be easily purified.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.14/873,321 filed Oct. 2, 2015, now U.S. Pat. No. 10,071,171, issued Sep.11, 2018, which is a divisional of U.S. application Ser. No. 13/055,406filed Apr. 14, 2011, now U.S. Pat. No. 9,636,420, issued May 2, 2017,which is a National Stage of International Application No.PCT/KR2009/004114, filed on Jul. 23, 2009, which claims the benefit ofpriority from Korean Patent Application No. KR 10-2008-0071766, filed onJul. 23, 2008, the contents of which are herein incorporated byreference in their entirety.

TECHNICAL FIELD

The present invention relates to a protein complex which allows the longacting activity of a physiologically active polypeptide with a dimericprotein. More particularly, the present invention relates to a proteincomplex in which a physiologically active polypeptide and a dimericprotein are linked to a non-peptidyl polymer having three functionalends (3-arm) via respective covalent bond, and a method for preparingthe same.

BACKGROUND ART

Due to low stability, polypeptides are generally apt to denature and bedegraded by proteinases and lose their activity. On the other hand,peptides are relatively small in size so that they are readily excretedthrough the kidney.

In order to maintain desired blood level concentrations and titersthereof, thus, protein medicines comprising polypeptides or peptides asactive ingredients need to be frequently administered. However, becauseprotein medicines are, for the most part, in a form suitable forinjections, the maintenance of physiologically active polypeptides orpeptides at appropriate blood levels requires frequent injections,causing significant pain to the patient. In order to overcome theseproblems, attempts have been made to provide maximum medicinal effectsby increasing the stability of protein medicines in the blood and bymaintaining blood medicine levels high for a long period of time. Theselong-lasting protein medicine agents are required not only to increasethe stability of the protein medicines and maintain sufficient titers ofthe medicines themselves, but also to not cause immune responses in thepatients.

Conventionally, highly soluble polymers such as polyethyleneglycol (PEG)are chemically grafted onto the surface of proteins with the aim ofstabilizing the proteins, preventing the contact of proteinases with theproteins, and suppressing the renal loss of small-size peptides. Graftedto specific or a variety of different sites on proteins, PEG is usefulfor the stabilization and hydrolysis prevention of proteins withoutcreating noteworthy side effects. In addition, grafted PEG increases themolecular weight of the proteins, thereby restraining renal loss of theproteins and maintaining the physiological activity of the proteins.

For example, WO 2006/076471 describes the use of B-type natriureticpeptide (BNP) in the treatment of congestive heart failure. BNP binds tonatriuretic peptide receptor A (NPR-A) to trigger the synthesis of cGMP,thereby reducing arterial blood pressures. When PEGylated, BNP isdescribed as elongating the physiological activity thereof for a longperiod of time. U.S. Pat. No. 6,924,264 also discloses an increase inthe active period of exendin-4 by grafting PEG onto a lysine residue.

In order to increase the physiological activity thereof, a medicinalpolypeptide is linked to both of the terminals of PEG to form abis-conjugate (U.S. Pat. No. 5,738,846). On the other hand, twodifferent medicinal proteins are linked to respective terminals of PEGto form a protein complex which have two different physiologicalactivities (WO 92/16221). However, no significance was found in theseprotein drugs in terms of activity maintenance.

Also, it was reported that a fusion protein in which G-CSF and humanalbumin were linked to one PEG increased in stability (Kinstler et al.,Pharmaceutical Research 12(12): 1883-1888, 1995). However, the modifieddrug with a G-CSF-PEG-albumin structure was found to increase inresidence time by only about four times, compared to natural drugsalone, and to be only slightly increased in serum half life. Thus, themodified drug is not practically applied as a lasting agent.

When coupled with PEG, peptides become so stable as to extend thepersistence thereof in vivo. However, when given high molecular weights,PEG makes the titer of the physiological active peptide significantlylow and decreases in reactivity with peptides, resulting in a low yield.

An alternative for increasing the stability of physiologically activeproteins in vivo takes recourse to gene recombination. A gene coding fora protein highly stable in the blood is linked to a gene coding for aphysiologically active protein of interest, followed by transformationinto animal cells which are then cultured to produce a fusion protein.

For example, a fusion protein in which albumin or a fragment thereof,known to be the most effective in stabilizing proteins thus far, isfused to a physiological active protein of interest has been reported(WO 93/15199 and 93/15200, EP Publication No. 413,622). Also, a fusionprotein of interferon alpha and albumin, produced from yeast by HumanGenome Sciences (trade name: Albuferon™) increased its serum half-lifefrom 5 hrs to 93 hrs, but suffers from the critical disadvantage ofbeing decreased in bioactivity to less than 5% of that of naiveinterferon (Osborn et al., J. Phar. Exp. Ther. 303(2): 540-548, 2002).

As for peptides, their modifications are mentioned in WO 02/46227 whichdiscloses that GLP-1, exendin-4 and analogs thereof are fused to humanserum albumin or immunoglobulin fragments (Fc) using geneticrecombination techniques and in U.S. Pat. No. 6,756,480 which disclosesfusion proteins of parathyroid hormone (PTH) or analogs thereof andimmunoglobulin fragments (Fc). These approaches can overcome lowpegylation yield and non-specificity, but are disadvantageous in thatserum half life is not significantly increased and in some cases, lowtiters result. Various peptide linkers are used to maximally increaseserum half life, but show the high possibility of causing immuneresponses. When given, a peptide having a disulfide bond, such as BNP,is highly likely to induce misfolding and thus is difficult to apply.

Other various fusion proteins are also known to be prepared by linkingthe Fc domain of immunoglobulin to interferon (Korean Patent PublicationNo. 2003-9464), interleukin-4 receptor, interleukin-7 receptor orerythropoietin receptor (Korean Patent No. 249572) through geneticrecombination. PCT Patent Publication No. WO 01/03737 discloses a fusionprotein in which a cytokine or a growth factor is linked through anoligopeptide linker to an Fc fragment of immunoglobulin. U.S. Pat. No.5,116,964 describes LHR (lymphocyte cell surface glycoprotein) or CD4protein which is fused to the amino or carboxy end of an immunoglobulinFc domain using a genetic recombination technique. Also, U.S. Pat. No.5,349,053 discloses a fusion protein in which IL-2 is linked to animmunoglobulin Fc domain. Many other Fc fusion proteins constructedusing genetic recombination techniques are disclosed, examples of whichinclude a fusion protein of an immunoglobulin Fc domain withinterferon-beta or a derivative thereof (PCT Patent Publication No. WO00/23472), an immunoglobulin Fc domain with an IL-5 receptor (U.S. Pat.No. 5,712,121), an immunoglobulin G4 Fc domain with interferon alpha(U.S. Pat. No. 5,723,125) and an immunoglobulin G2 Fc domain with a CD4protein (U.S. Pat. No. 6,451,313). On the other hand, U.S. Pat. No.5,605,690 teaches the use of a modified immunoglobulin Fc domain in theproduction of fusion proteins. For example, immunoglobulin Fc with aminoacid residues modified particularly to complement binding sites orreceptor binding sites is used to produce a TNFR-IgG1 Fc fusion proteinusing a genetic recombination method. Other fusion proteins of themodified immunoglobulin Fc domain which are produced using generecombination techniques are disclosed in U.S. Pat. Nos. 6,277,375,6,410,008 and 6,444,792.

Immunoglobulins function as antibodies, showing antibody-dependent cellcytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) and thesugar chains present in the immunoglobulin Fc domain are reported toplay an important role in ADCC and CDC (Burton D., Molec. Immun. 22,161-206, 1985). Immunoglobulins themselves, when free of sugar chains,are known to be similar in serum half life to the immunoglobulins havingsugar chains, but to have a 10- to 1000-fold decrease in complementbinding force and receptor binding force (Waldmann H., Eur. J. Immunol.23, 403-411, 1993; Morrison S., J. Immunol. 143, 2595-2601, 1989).

U.S. Pat. No. 6,660,843 discloses the fusion of an Fc domain with apeptide of interest through a linker and the production of the fusionprotein in E. coli using a gene recombination technique. For use inpreparing complexes, a linker allows the selection of the conjugationsites between two proteins of interest and the orientations thereof, andenables the production of complexes in the form of homogenous orheterogenous monomers, dimers or multimers. In addition, when using thismethod, the complexes can be produced at a lower cost than when usingmammal cells. In addition, the complexes may be produced in sugarchain-free forms. Because of the concomitant production of both theprotein of interest and the immunoglobulin Fc domain in E. coli,however, this method is difficult to apply to a target protein when thenative form of the target protein has a sugar chain. Taking advantage ofinclusion bodies, this method is highly apt to induce misfolding. In theFc fusion proteins produced using the genetic recombination techniques,fusion is possible only at specific sites, that is, an amino or carboxyterminus of the immunoglobulin Fc domain. The Fc fusion proteins areexpressed only in homogenous dimeric forms, but not in monomeric forms.Further, fusion is possible only between glycosylated proteins orbetween aglycosylated proteins, but impossible between glycosylatedproteins and aglycosylated proteins. If present, an amino acid sequencenewly formed as a result of the fusion may induce an immune response.Moreover, the linker may be sensitive to enzymatic degradation.

In the development of fusion proteins using immunoglobulin Fc domains,nowhere has an attempt been made to give complexes of target proteinswith human native Fc through a crosslinker in previous reports.Immunoglobulin Fc domains can be produced in mammal cells or E. coliusing genetic recombination techniques, but nowhere has an attempt beenmade to produce only native immunoglobulin Fc domains free of targetproteins at high yield and to apply them to lasting forms in previousreports. In addition, no attempts have been made to produce complexes ofthe recombinant immunoglobulin Fc with target proteins throughcrosslinkers.

As such, a variety of different methods have been performed in order toconjugate physiologically active polypeptides with polymers. Inconventional methods, polypeptides can be improved in stability, butwith significant reduction in activity, or can be improved in activityirrespective of stability. Therefore, there is still a need for a methodthat can increase the stability of protein medicines with a minimumreduction in modification-induced activity.

In this context, the present inventors developed a protein complex whichis improved in serum half life with high activity by linking animmunoglobulin and a physiologically active polypeptide respectively toopposite termini of a non-peptidyl linker as disclosed in Korean PatentNos. 10-0725315 and 10-0775343, which are incorporated by reference intheir entirety.

A protein complex in which an immunoglobulin and a physiologicallyactive polypeptide are respectively linked to opposite termini of anon-peptidyl polymer is conventionally prepared by linking anon-peptidyl polymer preferentially with a physiologically activepolypeptide and then with an immunoglobulin Fc domain. However, thisconventional preparation method produces lots of undesirable impurities,as well, resulting in losing a large quantity of the physiologicallyactive polypeptide. That is, the conventional method is economicallyunfavorable upon the industrial application thereof and the resultingcomplex must be purified in a somewhat complicated manner. In the casewhere the physiologically active polypeptide is in the form of a dimer,it produces a bridge form with a non-peptidyl polymer at both termini sothat it cannot complex with an immunoglobulin Fc or can complex but atvery low yield. On the other hand, when an immunoglobulin Fc domain isfirst linked to a non-peptidyl polymer, similar problems occur as well.Because an immunoglobulin Fc is a homo-dimer with two N-termini in closevicinity to each other, respective links are formed between the twoN-termini of the immunoglobulin Fc and the opposite termini of thenon-peptidyl polymer to produce a bridge form, so that no functionalends remain to be reacted with the physiologically active polypeptide.Accordingly, the production yield significantly decreases.

DISCLOSURE Technical Problem

Leading to the present invention, intensive and thorough research intoprotein complexes, conducted by the present inventors, aiming toovercome the problems encountered in the prior art, resulted in thefinding that the use of 3-arm non-peptidyl polymer as a linker inpreparing a protein complex composed of a dimeric protein and aphysiologically active polypeptide prevents the loss of thephysiologically active polypeptide to significantly increase theproduction yield, allows the complex to be purified with a simplemethod, and gives structural stability to the protein complex to extendthe serum half life while at the same time maintaining the biologicalactivity thereof.

Technical Solution

It is therefore an object of the present invention to provide a proteincomplex in which a physiologically active polypeptide, a 3-armnon-peptidyl polymer, and a dimeric protein are linked via covalentbonds, and a method for preparing the same.

It is another object of the present invention to provide a sustainedprotein drug agent comprising a protein complex which extends the serumhalf life of a physiologically active polypeptide while maintaining thebiological activity thereof.

Advantageous Effects

Having a structure in which a physiologically active polypeptide and adimeric protein are linked to a 3-arm non-peptidyl polymer via covalentbonds, the protein complex of the present invention can maintain a highblood concentration of the active polypeptide over a long period of timeproducing stable medicinal effects.

In addition, the method for preparing the protein complex in accordancewith the present invention can greatly reduce the amount ofphysiologically active polypeptide required by the conventional methodusing a 2-arm non-peptidyl polymer and enjoys, over the conventionalmethod, the advantage of introducing more simple purification methodsand significantly increasing the production yield. Especially,persistent protein complexes with dimeric, physiologically activepolypeptides were preferably prepared using the method of presentinvention.

DESCRIPTION OF DRAWINGS

FIG. 1 shows representative figure of a protein complex using anon-peptidyl polymer having three functional ends (3-arm).

FIG. 2 shows in vivo efficacies of the immunoglobulin Fc-3 armPEG-Octreotide(N) complex in plots (HM11760B: immunoglobulin Fc-3 armPEG-Octreotide(N) complex, Sandostatin-LAR: a sustained releaseformulation of octreotide).

FIG. 3 shows in vivo efficacy of the immunoglobulin Fc-3 arm PEG-FSH(N)complex (HM12160A: immunoglobulin Fc-2 arm PEG-FSH(N) complex, HM12160B:immunoglobulin Fc-3 arm PEG-FSH(N) complex).

BEST MODE

In accordance with an aspect thereof, the present invention is directedto a protein complex in which a physiologically active polypeptide and adimeric protein are covalently linked to a 3-arm non-peptidyl polymer.

As used herein, the term “protein complex” or “complex” is intended torefer to a structure composed of at least one physiologically activepolypeptide, at least one 3-arm non-peptidyl polymer and at least onedimeric protein, with interconnection via covalent bonds among them. Inorder to differentiate itself from a “complex”, the term “conjugate” isused herein to refer to a structure in which only pairs of thephysiologically active polypeptide, the non-peptidyl polymer and thedimeric protein are interconnected via a covalent bond.

The protein complex of the present invention is a protein drug which ismodified to increase the persistence thereof in vivo and minimallyreduce the biological activity thereof. The present invention featuresthe use of a 3-arm non-peptidyl polymer to connect a physiologicallyactive polypeptide and a dimeric protein therethrough to form a proteincomplex, thereby allowing the application of a preparing method by whichthe loss of the physiologically active polypeptide can be prevented andthe protein complex can be so structurally stable as to be purifiedsimply.

As used herein, the term “dimeric protein” means a protein with twoN-termini. Preferred is an immunoglobulin Fc domain which can be used asa carrier. Physiologically active homodimers or heterodimers are alsoincluded within the scope of the dimeric protein.

An immunoglobulin Fc domain is stable enough to be used as a carrier fora drug because it is a biodegradable polypeptide which is metabolized invivo. In addition, thanks to relatively small molecular weights, theimmunoglobulin Fc domain has advantages over total immunoglobulinmolecules in terms of the preparation, purification and yield of thecomplex. Further, because it is free of Fab that is highly different inamino acid sequence from one antibody to another, Fc strongly promotesthe homogeneity of the complex and is expected to reduce the inductionof antigenicity.

The term “immunoglobulin Fc domain”, as used herein, is intended toindicate heavy chain constant domain 2 (C_(H)2) and heavy chain constantdomain 3 (C_(H)3) which is free of heavy and light chain variabledomains, heavy chain constant domain 1 (C_(H)1) and light chain constantdomain 1 (C_(L)1) and may comprise a hinge region. The immunoglobulin Fcdomain of the present invention may be an extended Fc domain whichfurther comprises a part or total heavy chain constant domain 1 (C_(H)1)and/or light chain constant domain 1 (C_(L)1) and is free of the heavyand light chain variable domains if it guarantees an effectsubstantially the same as or higher than that of the native form.Alternatively, the Fc domain may be a truncated form of C_(H)2 and/orC_(H)3 which lacks a significant part of the corresponding amino acidsequence. To sum up, the immunoglobulin Fc domain of the presentinvention may be 1) C_(H)1 domain, C_(H)2 domain, C_(H)3 domain andC_(H)4 domain, 2) C_(H)1 domain and C_(H)2 domain, 3) C_(H)1 domain andC_(H)3 domain, 4) C_(H)2 domain and C_(H)3 domain, 5) a combination ofone or more domains and an immunoglobulin hinge region (or a part ofhinge) or 6) a dimer composed of each light chain constant domain and alight chain constant domain.

In addition, the term “immunoglobulin Fc domain”, as used herein, isintended to cover not only native amino acid sequences but also mutantsthereof. The amino acid sequence mutant means an amino acid sequencedifferent from the native sequence by deletion, insertion,non-conservative or conservative substitution of one or more amino acidresidues or combinations thereof. For example, the amino acid residuesat positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 of IgGFc, which are known to play an important role in antibody binding, maybe modified so as to be used as suitable binding sites. In addition,possible are various mutants which, for example, lack a residue forminga disulfide bond or several N-terminal amino acids of the native Fc, orhave an additional methionine residue at the N terminus of the nativeFc. Further, effector functions may be eliminated by removing acomplement binding motif, e.g., C1q binding motif, or an ADCC(antibody-dependent cell mediated cytotoxicity) motif. Reference may bemade to WO 97/34631 and WO 96/32478 concerning the preparation of aminoacid sequence mutants of immunoglobulin Fc domains.

Amino acid substitutions which do not alter the activity of nativeproteins or peptides as a whole are known in the art (H. Neurath, R. L.Hill, The Proteins, Academic Press, New York, 1979). Most typicalsubstitutions occur between Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly,Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn,Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly. If necessary, the amino acidsmay undergo a modification, such as phosphorylation, sulfation,acrylation, glycosylation, methylation, farnesylation, acetylation,amidation, etc.

The above-described Fc mutants are preferably functional equivalents totheir natural forms, thus being similar in biological activity, with animprovement in structural stability against heat and pH.

The Fc domain may be a native form isolated from humans and otheranimals including cows, goats, pigs, mice, rabbits, hamsters, rats andguinea pigs, or may be a recombinant or a derivative thereof, obtainedfrom transformed animal cells or microorganisms. In the former case,total immunoglobulin is isolated from humans or animals, followed byprotease treatment. When treated with papain, the total immunoglobulinis divided into Fab and Fc. Pepsin cleaves total immunoglobulin intopF′c and F(ab)2. From these fragments, Fc or pF′c can be separated usingsize-exclusion chromatography. Preferred is a recombinant immunoglobulinFc domain derived from the human Fc domain in microorganisms.

The immunoglobulin Fc domain useful in the present invention may have asugar chain less than, equal to, or longer in length than the native Fcdomain, or may not have sugar chains. The addition, reduction or removalof the immunoglobulin Fc sugar chain may be achieved using a typicaltechnique, such as a chemical technique, an enzymatic technique or agenetic recombination technique using microorganism. A deglycosylatedimmunoglobulin Fc domain is significantly reduced in the binding forceof the complement (C1q) and has little or no antibody-dependentcell-mediated cytotoxicity or complement-dependent cytotoxicity, andthus induces no unnecessary immune responses. In this context,deglycosylated or aglycosylated immunoglobulin Fc domains preferentiallycoincide with the intended function as drug carriers.

As used herein, the term “deglycosylation” refers to the enzymaticremoval of a sugar chain from the native Fc. The term “aglycosylation”refers to the absence of sugar chains in the Fc domain because it isproduced in eucaryotes and preferably in E. coli.

The immunoglobulin Fc domain may originate from animals includinghumans, cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigswith a preference for human origin. In addition, the immunoglobulin Fcdomain useful in the present invention may be derived from among IgG,IgA, IgD, IgE, IgM and combinations thereof or hybrids thereof.Preferably, it is derived from IgG or IgM, which are more abundant thanthe other types of immunoglobulin, and most preferably from IgG which isknown to extend the serum half life of ligand binding proteins.

Also, the immunoglobulin Fc domain may be in the form of dimers ormultimers (combinations of immunoglobulin Fc), each comprisingglycosylated immunoglobulins composed of domains of the same origin.

The term “combination”, as used herein, means that polypeptides encodingsingle-chain immunoglobulin Fc fragments of the same origin are linkedto a single-chain polypeptide of a different origin to form a dimer ormultimer. That is, a dimer or a multimer may be prepared by combiningtwo or more fragments selected from among the Fc fragments of IgG Fc,IgA Fc, IgM Fc, IgD Fc and IgE Fc.

The term “hybrid”, as used herein, means that sequences encoding two ormore immunoglobulin Fc fragments of different origins are present in asingle-chain immunoglobulin Fc fragment. In the present invention,various hybrid forms are possible. For example, the Fc domain iscomposed of one to four different domains selected from C_(H)1, C_(H)2,C_(H)3 and C_(H)4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc and maycomprise a hinge region.

IgG is also further divided into the subclasses of IgG1, IgG2, IgG3 andIgG4 and their combinations or hybrids are permitted in the presentinvention. Preferable is an Fc domain of IgG2 or IgG4, with the highestpreference being given for an Fc domain of IgG4 free of effectorfunctions such as complement-dependent cytotoxicity (CDC).

Accordingly, an aglycosylated Fc domain of human IgG4 is the most highlypreferred drug carrier. The Fc domain of human origin is advantageousover that of non-human origin because the latter may act as an antigenin the body, inducing the production of antibodies thereto.

The present invention features the linkage of a protein drug to adimeric protein via a non-peptidyl polymer.

In the present invention, the non-peptidyl polymer means a biocompatiblepolymer composed of two or more repeating units which are linked to eachother via a covalent bond other than a peptide bond.

Conventional peptidyl linkers used in the fusion proteins preparedthrough in-frame fusion suffer from the disadvantage of being easilycleaved in vivo by proteinases to fail to guarantee the serum half lifeof the active drug as long as expected when the carrier remains intact.In contrast, the polymer of the present invention is resistant toenzymatic degradation, maintaining the serum half life of the drug aslong as that guaranteed when the carrier remains intact. So long as itacts as mentioned above, that is, is resistant in vivo to proteinases,any polymer may be used in the present invention with no limitationsimparted thereto. The non-peptidyl polymer useful in the presentinvention ranges in molecular weight from 1 to 100 kDa and preferablyfrom 1 to 20 kDa. The non-peptidyl polymer of the present inventionwhich is conjugated with an immunoglobulin Fc domain may be a polymer ora combination of different polymers.

Examples of the non-peptidyl polymer useful in the present inventioninclude biodegradable polymers such as polyethylene glycol,polypropylene glycol, a copolymer of ethylene glycol and propyleneglycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide,dextran, polyvinyl ethyl ether, biodegradable polymers such as PLA(polylactic acid) and PLGA (polylactic-glycolic acid), lipopolymers,chitins, hyaluronic acid and combinations thereof, with a preference forpolyethylene glycol. Also, their derivatives which are known in the artor which can be readily prepared using a typical technique fall withinthe scope of the present invention.

The non-peptidyl polymers used in the present invention have functionalgroups to which the immunoglobulin Fc domain and the protein drug canbind.

In contrast to the prior method for preparing protein complex using anon-peptidyl polymer having two functional ends (2-arm) (Korean PatentNo. 10-0725315), the present invention employs a 3-armnon-peptidylpolymer. Different from the conventional non-peptidyl polymer which hasone functional end and branches from a core molecule, the non-peptidylpolymer useful in the present invention has three functional ends inwhich two are responsible for the formation of covalent bonds with adimeric protein while the other one is covalently linked to aphysiologically active polypeptide.

In greater detail, because an immunoglobulin Fc is a homo-dimer with twoN-termini in close vicinity to each other, respective links are formedbetween the two N-termini of the immunoglobulin Fc and the oppositetermini of the non-peptidyl polymer. Thus, when the 2-arm non-peptidylpolymer first forms covalent bonds with the immunoglobulin Fc domain,there are no functional ends left that can be reacted with thephysiologically active polypeptide, resulting in a significant decreasein production yield. Thus, the physiologically active polypeptide, priorto the 2-arm non-peptidyl polymer, is reacted with the immunoglobulin Fcdomain. However, when the physiologically active polypeptide is reactedin advance, lots of undesirable impurities are produced, thus givingrise to the loss of the physiologically active polypeptide. In contrast,it is possible to react the 3-arm non-peptidyl polymer withimmunoglobulin Fc domain before reaction with the physiologically activepolypeptide because two of its three functional ends are responsible fortwo N-termini of the immunoglobulin Fc while the other functional endthereof can cover the physiologically active polypeptide. Hence, theprotein complex can be produced in high yield. In practice, theproduction yield is found to increase two ˜nine times more than when2-arm non-peptidyl polymers are used.

The three terminal functional groups of the non-peptidyl polymer canbind to N-terminal, free lysine, histidine or cysteine residues of theimmunoglobulin Fc domain and the physiologically active polypeptide.

Preferably, the three terminal functional groups of the non-peptidylpolymer are selected from among aldehyde groups, propionaldehyde groups,butyl aldehyde groups, maleimide groups and succinimide derivatives.Examples of the succinimide derivatives useful in the present inventioninclude succinimidyl propionate, hydroxy succinimidyl, succinimidylcarboxymethyl and succinimidyl carbonate. Preferred are aldehyde groups.When present on the three termini of the non-peptidyl polymer, reactivealdehyde groups can minimize non-specific reactions and are effective informing bonds with both the physiologically active polypeptide and thedimeric protein. Further, the final product formed by reductivealkylation with aldehyde is far more stable than is the product formedwith amide bonds. Generally, aldehyde functional groups selectivelyreact with amino ends at a low pH while forming a covalent bond with alysine residue at high pH, e.g., pH 9.0. The three terminal functionalgroups of the non-peptidyl polymer may be the same or different fromeach other.

In accordance with the present invention, the conjugate of the dimericprotein with the non-peptidyl polymer is linked to a physiologicallyactive polypeptide to form a protein complex.

Herein, the term “physiologically active polypeptide”, “physiologicallyactive protein”, “active protein”, “active polypeptide” or “proteindrug” means a polypeptide, a peptide or a protein having some kind ofantagonistic activity to a physiological event in vivo and these termsmay be used interchangeably.

The physiologically active polypeptides applicable to the proteincomplex of the present invention may be exemplified by hormones,cytokines, interleukins, interleukin-binding proteins, enzymes,antibodies, growth factors, transcription factors, blood factors,vaccines, structural proteins, ligand proteins, receptors, cell surfaceantigens, receptor antagonists, and derivatives or analogs thereof.

Concrete examples of the physiologically active polypeptides useful inthe present invention include human growth hormones, growth hormonereleasing hormones, growth hormone releasing peptides, interferons andinterferon receptors (e.g., interferon-α, -β and -γ, soluble type Iinterferon receptors), colony-stimulating factors, glucagon-likepeptides (GLP-1, etc.), exendin-4 peptides, ANP (atrial natriureticpeptide), BNP (brain natriuretic peptide), CNP (c-type natriureticpeptide), DNP (dendroaspis natriuretic peptide), G protein-coupledreceptors, interleukins (e.g., interleukin-1, -2, -3, -4, -5, -6, -7,-8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, -21, -22,-23, -24, -25, -26, -27, -28, -29, -30) and interleukin receptors (e.g.,IL-1 receptor, IL-4 receptor, etc.), enzymes (e.g., glucocerebrosidase,iduronate-2-sulfatase, α-galactosidase-A, α-L-iduronidase,butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase,lipase, uricase, platelet-activating factor acetylhydrolase,neutralendopeptidase, myeloperoxidase, etc.), interleukin- andcytokine-binding proteins (e.g., IL-18 bp, TNF-binding protein, etc.),macrophage activating factors, macrophage peptides, B-cell factors,T-cell factors, Protein A, allergy inhibitors, cell necrosisglycoproteins, immunotoxins, lymphotoxins, tumor necrosis factor, tumorsuppressors, transforming growth factor, alpha-1 anti-trypsin, albumin,α-lactalbumin, apolipoprotein-E, erythropoietin, glycosylatederythropoietin, angiopoeitins, hemoglobin, thrombin, thrombin receptorsactivating peptides, thrombomodulin, blood factor VII, VIIa, VIII, IXand XIII, plasminogen activators, fibrin-binding peptides, urokinase,streptokinase, hirudin, Protein C, C-reactive protein, renin inhibitor,colagenase inhibitor, superoxide dismutase, leptin, platelet-derivedgrowth factor, epithelial growth factor, epidermal growth factor,angiostatin, angiotensin, bone growth factor, bone stimulating protein,calcitonin, insulin, somatostatin, octreotide (somatostatin agonist),atriopeptin, cartilage inducing factor, elcatonin, connective tissueactivating factor, tissue factor pathway inhibitor, follicle stimulatinghormone, luteinizing hormone, luteinizing hormone releasing hormone,nerve growth factors (e.g., nerve growth factor, cilliary neurotrophicfactor, axogenesis factor-1, glucagon-like-peptides (GLP-1), exendin-4peptides, brain-natriuretic peptide, glial derived neurotrophic factor,netrin, neurophil inhibitor factor, neurturin, etc.), parathyroidhormone, relaxin, secretin, somatomedin, insulin-like growth factor,adrenocortical hormone, glucagon, cholecystokinin, pancreaticpolypeptide, gastrin releasing peptide, corticotropin releasing factor,thyroid stimulating hormone, autotaxin, lactoferrin, myostatin,receptors (e.g., TNFR(P75), TNFR(P55), IL-1 receptor, VEGF receptor,B-cell activator receptor, etc.), receptor antagonists (e.g., IL1-Ra,etc.), cell surface antigens (e.g., CD 2, 3, 4, 5, 7, 11a, 11b, 18, 19,20, 23, 25, 33, 38, 40, 45, 69, etc.), monoclonal antibodies, polyclonalantibodies, antibody fragments (e.g., scFv, Fab, Fab′ F(ab′)2 and Fd),and virus derived vaccine antigents. Preferably, the physiologicallyactive polypeptide is selected from among human growth hormones,interferon-alpha, interferon-beta, granulocyte colony stimulatingfactor, erythropoietin, exendin-4, imidazole acetyl exendin-4 peptide(exendin-4 agonist), calcitonin, octreotide (somatostatin agonist), BNPand Fab′.

Disadvantageously, these protein drugs cannot maintain their biologicalactivity in vivo over a long time because they are highly apt todenature or are readily degraded with proteinases. However, the complexof the present invention in which a dimeric protein and a polypeptideare conjugated via a non-peptidyl polymer increases both the structuralstability and half clearance time of the drug. The reduction of thebiological activity of the polypeptide because of conjugation with thedimeric protein is very insignificant, compared to that generated inconventional complexes. Therefore, the complex of the polypeptide andthe immunoglobulin Fc domain in accordance with the present invention ischaracterized by having a significantly improved bioavailability,compared to that of conventional polypeptide drug agents.

In accordance with another aspect thereof, the present inventionpertains to a method for preparing a protein complex in which a dimericprotein is conjugated with a physiologically active polypeptide via a3-arm, non-peptidyl polymer, comprising: (1) covalently linking two armsof the 3-arm non-peptidyl polymer to opposite N-terminal amino groups ofthe dimeric protein to form a conjugate (2) isolating from the reactionmixture of step (1) the conjugate in which the dimeric protein iscovalently linked at N-termini thereof with the non-peptidyl polymer and(3) covalently linking the physiologically active polypeptide to onefree arm of the non-peptidyl polymer of the isolated conjugate.

In a preferred embodiment, the dimeric protein of step (1) is animmunoglobulin Fc domain. In another preferred embodiment, the threearms of the non-peptidyl polymer of step (1) have respective aldehydefunctional groups at termini thereof. More preferably, the non-peptidylpolymer is linked to the N-terminal amino group of the physiologicallyactive polypeptide at pH 6.0.

In step (1), the dimeric protein is reacted at a molar ratio of from 1:2to 1:5 with the non-peptidyl polymer. In step (3), the molar ratio ofthe conjugate isolated by step (2): the physiologically activepolypeptide preferably ranges from 1:0.5 to 1:0.05.

The reactions in steps (1) and (3) depend on the three terminal groupsof the non-peptidyl polymer. If required, the reactions may be conductedin the presence of a reducing agent. Preferred examples of the reducingagent include sodium cyanoborohydride (NaCNBH₃), sodium borohydride,dimethylamine borate and pyridine borate.

The linkage between the immunoglobulin Fc domain and the physiologicallyactive polypeptide is achieved not by fusion based on geneticrecombination, but via a covalent bond.

In accordance with a further aspect thereof, the present inventionpertains to a pharmaceutical composition comprising the protein complexof the present invention and a pharmaceutically acceptable vehicle,which shows an improvement in the in-vivo sustainability of aphysiologically active polypeptide.

Via an appropriate route, the pharmaceutical composition of the presentinvention must be introduced into a tissue or organ of interest. So longas it is delivered to the targeted tissue, any route can be used foradministration of the pharmaceutical composition of the presentinvention. For example, the administration may be carried out viaintraperitoneal, intravenous, intramuscular, subcutaneous, intradermal,oral, local, intranasal, intrapulmonary, and intrarectal routes, but isnot limited thereto. Preferably, the pharmaceutical composition of thepresent invention is administered in an injectable form. Also, thepharmaceutical composition may be administered with the aid of a devicethrough which the active material is delivered to targeted cells.

The pharmaceutical composition based on the complex of the presentinvention may further comprise a pharmaceutically acceptable vehicle.For use as a pharmaceutically acceptable vehicle in oral dosage forms, abinder, a lubricant, a disintegrant, an excipient, a solubilizer, adispersant, a stabilizer, a suspending agent, a colorant, and/or afragment are selected. When the pharmaceutical composition of thepresent invention is formulated into injections, a buffer, apreservative, a pain relieving agent, a solubilizer, an isotonic, and astabilizer may be used alone or in combination. For local application, abase, an excipient, a lubricant, and a preservative may be used. Forpractical use, the pharmaceutical composition of the present inventionmay be formulated in combination with the pharmaceutically acceptablevehicle into various dosage forms. For dosage forms, for example, it canbe formulated into tablets, troches, capsules, elixir, suspensions,syrups, wafers, etc. As concerns injections, they may be in the form ofsingle dose ampoules or multiple doses. Other available forms includesolutions, suspensions, pills, powders, capsules, and sustained releaseagents. Examples of vehicles, excipients and diluents useful in theformulation include lactose, d-lactose, dextrose, sucrose, sorbitol,mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate,gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinyl pyrrolidone, water,methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearateand mineral oil. Also, a filler, an anti-agglomerating agent, alubricant, a fragrant, an emulsifier, and a preservative are useful.

The pharmaceutically effective amount of the protein complex of thepresent invention varies depending on the kind of diseases to betreated, the route and frequency of administration, the age, gender andweight of the patient, and the severity of disease, as well as the kindof the physiologically active polypeptides. The superiority of theprotein complex in in vivo persistency and titer makes it possible toreduce the administration dose and frequency of the pharmaceuticalcomposition of the present invention.

As disclosed in Korean Patent Nos. 10-0725315 and 10-0775343, a proteincomplex is provided in which an immunoglobulin constant domain and aphysiologically active polypeptide are respectively linked to oppositetermini of a non-peptidyl polymer. This protein complex is prepared bylinking the non-peptidyl polymer with the physiologically activepolypeptide to form a conjugate and then with the immunoglobulin Fc. Thepreparation process is disadvantageous in that the physiologicallyactive polypeptide is lost in a large quantity during the preparation.In contrast, the use of a 3-arm non-peptidyl polymer in the preparationof a protein complex can exceptionally reduce the loss of thephysiologically active polypeptide. Further, the protein complex can beisolated using a simple purification method.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as limiting the present invention.

MODE FOR INVENTION Example 1: Preparation of Immunoglobulin Fc-3 ArmPEG-Octreotide(N) Complex

For pegylation of an immunoglobulin Fc at its N-terminus, 10 mg/ml ofimmunoglobulin Fc was reacted at a molar ratio of 1:2 with 5K PropionALD(3) PEG (PEG with three propionaldehyde groups, NOF, Japan) at 4° C. for4.5 hrs. The reaction was conducted in 100 mM potassium phosphatebuffer, pH 6.0, in the presence of 20 mM SCB (NaCNBH₃) as a reducingagent. Using SOURCE Q (XK 16 ml, GE Healthcare), mono-pegylatedimmunoglobulin Fc was purified from the reaction mixture. Then,octreotide was reacted at a molar ratio of 1:2 with the immunoglobulinFc-5K PEG conjugate at 4° C. for 20 hrs, with total proteinconcentration set at 25 mg/ml. This coupling reaction was carried out in100 mM Potassium phosphate, pH 6.0, in the presence of the reducingagent 20 mM SCB. The coupling reaction mixture was purified through anSP HP purification column. Using a 10 mM sodium phosphate buffer (pH5.4) as a binding buffer, the immunoglobulin Fc-5K PEG conjugate whichdid not undergo the coupling reaction was not attached to an SP HPcolumn (XK 16 ml, GE Healthcare), but was separated by loading throughthe column, while the immunoglobulin-PEG-Octreotide (HM11760B) wasattached slightly to the column and then eluted with a salt gradient of1M NaCl.

Column: SP HP (XK 16 ml, GE Healthcare)

Flow rate: 2.0 ml/min

Gradient: A 0→20% 60 min B (A: 10 mM Na—P pH5.4, B: A+1M NaCl)

Example 2: Preparation of Immunoglobulin Fc-3 Arm PEG-Calcitonin (N)Complex

Immunoglobulin-Fc was reacted at its N-termini with 5K PropionALD (3)PEG in the same manner as in Example 1, after which only themono-pegylated immunoglobulin Fc was purified and coupled withcalcitonin. In this regard, calcitonin was reacted at a molar ratio of1:2 with the immunoglobulin Fc-5K PEG conjugate at 4° C. for 20 hrs,with total protein concentration set at 25 mg/ml. For use as a medium inthis coupling reaction, 100 mM potassium phosphate buffer, pH 6.0, wassupplemented with the reducing agent 20 mM SCB (NaCNBH₃). The couplingreaction mixture was purified through an SP HP purification column.First, an SP HP column (XK 16 ml, GE Healthcare) was used to remove theimmunoglobulin Fc-5K PEG conjugate which remained uncoupled withcalcitonin. Using a 10 mM sodium phosphate buffer (pH 5.4) as a bindingbuffer, the immunoglobulin Fc-5K PEG conjugate which did not undergo thecoupling reaction was not attached to an SP HP column (XK 16 ml, GEHealthcare), but was separated by loading through the column, while theimmunoglobulin-PEG-calcitonin was attached slightly to the column andthen eluted with a salt gradient of 1M NaCl.

Column: SP HP (XK16 ml, GE Healthcare)

Flow rate: 2.0 ml/min

Gradient: A 0→20% 60 min B (A: 10 mM Na—P pH5.4, B: A+1M NaCl)

Example 3: Comparison of Production Yield and Purification ProcessSimplicity Between 3 Arm PEG Complex (EXAMPLE 1 and 2) and 2 Arm PEGComplex

Production yield of 3 arm PEG complex and 2 arm PEG complex was comparedusing the immunoglobulin Fc-3 arm PEG-Octreotide(N) complex and theimmunoglobulin Fc-3 arm PEG-Calcitonin (N) complex prepared by EXAMPLE 1and 2, respectively (table 1).

2 arm PEG complex as a control group was prepared by linking Octreotideor Calcitonin as a physiologically active polypeptide to theimmunoglobulin Fc region via 2 arm PEG (PEG with two propionaldehydegroups, IDB, South Korea) as a linker. Particularly, a 2-arm PEG wascoupled with Octreotide or Calcitoninand then with an immunoglobulin Fcdomain via a covalent bond (KR10-0725315).

TABLE 1 Active Pharmaceutical Production Yield Ingredient (API) PEG form(API basis) Octreotide 2 arm  9% 3 arm 33% Calcitonin 2 arm 18% 3 arm30%

The processes for purifying the complex from the coupling reactionmixture were analyzed for simplicity and primary column size and theresults are summarized in Table 2, below.

TABLE 2 Size of API PEG form Purification Process Primary ColumnOctreotide 2 arm Source Q −> Source ISO 5 3 arm SP HP 1 Calcitonin 2 armSource Q −> Source ISO 4 3 arm SP HP 1

When using the 2 arm PEG complex, all the immunoglobulin Fc whichremained uncoupled was attached to the column so as to separate itselffrom the immunoglobulin Fc-2 arm PEG-Octreotide or the immunoglobulinFc-2 arm PEG-Calcitonin. In contrast, when using the 3 arm PEG complex,separation between the immunoglobulin Fc-5K PEG conjugate which remaineduncoupled with the physiologically active polypeptide and theimmunoglobulin Fc-3 arm PEG-Octreotide or the immunoglobulin Fc-3 armPEG-Calcitonin was achieved in such a manner that the immunoglobulinFc-5K PEG conjugate was removed by loading through the column while theimmunoglobulin Fc-3 arm PEG-Octreotide or the immunoglobulinFc-3 armPEG-Calcitonin was attached to the column. Thus, not only was thepurification process reduced from two steps to one, but also the columnwas ⅕ reduced in size.

Example 4: Preparation of Immunoglobulin Fc-3 Arm PEG-FSH(N) Complex

Immunoglobulin-Fc was reacted at its N-termini with 5K PropionALD (3)PEG in the same manner as in Example 1, after which only themono-pegylated immunoglobulin Fc was purified and coupled with FSH. Inthis regard, FSH was reacted at a molar ratio of 1:15 with theimmunoglobulin Fc-5K PEG conjugate at 4° C. for 20 hrs, with totalprotein concentration set at 40 mg/ml. For use as a medium in thiscoupling reaction, 100 mM potassium phosphate buffer, pH 6.0, wassupplemented with the reducing agent 20 mM SCB. The coupling reactionmixture was purified through two purification columns. First, a Blue HPcolumn (Hitrap 5 ml, GE Healthcare) was used to remove theimmunoglobulin Fc-5K PEG conjugate which remained uncoupled with FSH.Subsequently, multi-polymers in which two or more immunoglobulin Fc-5KPEG conjugate were coupled with FSH were removed through Resource Iso (1ml, GE Healthcare) on the basis of hydrophobicity.

Column: Blue HP (Hitrap 5 ml, GE Healthcare)

Flow rate: 3.0 ml/min

Gradient: A 0→100% 20 min B

A: 50 mM Gly-NaOH+0.2M KCl

B: 50 mM Gly-NaOH+2.5M KCl

Column: Resource ISO (1 ml, GE Healthcare)

A 0→100% 90 min B (A: 20 mM Tris pH 7.5, B: A+1.3M A.S)

Example 5: Preparation of Immunoglobulin Fc-3 Arm PEG-Insulin (N)Complex

For pegylation of an immunoglobulin Fc at its N-terminus, 10 mg/ml ofimmunoglobulin Fc was reacted at a molar ratio of 1:2 with 5K PEGPropionALD (3) PEG (PEG with three propionaldehyde groups, NOF, Japan)at 4° C. for 4.5 hrs. The reaction was conducted in a 100 mM potassiumphosphate buffer, pH 6.0, in the presence of 20 mM SCB (NaCNBH₃) as areducing agent. Using SOURCE Q (LRC25 85 ml, GE Healthcare),mono-pegylated immunoglobulin Fc was purified from the reaction mixture.Then, insulin was reacted at a molar ratio of 1:4 with theimmunoglobulin Fc-5K PEG conjugate at 4° C. for 19.5 hrs. For use as amedium for this coupling reaction, 100 mM potassium phosphate buffer, pH6.0, was supplemented with the reducing agent 20 mM SCB. The couplingreaction mixture was purified through two purification columns. First, aSOURCE Q column (LRC25 85 ml, GE Healthcare) was used to remove theimmunoglobulin Fc-5K PEG conjugate which remained uncoupled withinsulin. A salt gradient of 1 M NaCl in 20 mM Tris (pH7.5) allowed theimmunoglobulin Fc-5K PEG conjugate to be eluted first due to relativelyweak binding force, immediately followed by the elution ofimmunoglobulin Fc-PEG-Insulin. Through this primary purification,immunoglobulin Fc-5K PEG conjugate was removed, but multi-polymers ofFc-5K PEG and Insulin were not separated completely. Secondarypurification was thus conducted on the basis of difference in molecularweight between the complex and the multi-polymer using Sephacryl S-300(GE Healthcare) column. At the same time, the immunoglobulinFc-PEG-Insulin complex was formulated. High-molecular weightmulti-polymers of immunoglobulin Fc-5K PEG and insulin were firsteluted, followed by the immunoglobulin Fc-PEG-Insulin complex.

Column: Source Q (LRC25 85 ml, GE Healthcare)

Flow rate: 8.0 ml/min

Gradient: A 0→25% 100 min B (A: 20 mM Tris pH7.5, B: A+1M NaCl)

Column: Sephacryl S-300 (HiPrep 120 ml, GE Healthcare)

Flow rate: 0.6 ml/min

Example 6: Comparison of Production Yield Between 3 Arm PEG Complex(Example 4 and 5) and 2 Arm PEG Complex

Production yield of 3 arm PEG complex and 2 arm PEG complex was comparedusing the immunoglobulin Fc-3 arm PEG-FSH(N) complex and theimmunoglobulin Fc-3 arm PEG-Insulin (N) complex prepared by EXAMPLE 4and 5, respectively (table 3).

When using the conventional preparation process using 2 arm PEG, dimericphysiologically active polypeptides, such as FSH (Mw. Ca. 40,000 Da) andinsulin (Mw. 5,807), occupied both the termini of the non-peptidylpolymer so that the polymer could not form a covalent bond with theimmunoglobulin Fc domain which provided persistence in vivo. Thisphenomenon was more conspicuous with a dimeric physiologically activepolypeptide of small molecular weight, such as insulin.

Accordingly, persistent protein complexes with dimeric physiologicallyactive polypeptides were preferably prepared using the 3 arm PEG.

Differences in the production yield of FSH and Insulin according topreparation process using 2 arm PEG and 3 arm PEG are summarized inTable 3, below.

TABLE 3 Active Pharmaceutical Production Yield Ingredient (API) PEG form(API basis) FSH 2 arm PEG 10% 3 arm PEG 32% Insulin 2 arm PEG  3% 3 armPEG 27%

Example 7: Preparation of Immunoglobulin Fc-PEG-FacVIIa (N) Complex

For pegylation of an immunoglobulin Fc at its N-terminus, 6 mg/ml ofimmunoglobulin Fc was reacted at a molar ratio of 1:2 with 5K PropionALD(3) PEG (PEG with three propionaldehyde groups, NOF, Japan) at 4° C. for4.5 hrs. The reaction was conducted in a 100 mM potassium phosphatebuffer, pH 6.0, in the presence of 20 mM SCB (NaCNBH₃) as a reducingagent. Using SOURCE Q (LRC25 85 ml, GE Healthcare), mono-pegylatedimmunoglobulin Fc was purified from the reaction mixture. Then, FVIIawas reacted at a molar ratio of 1:9 with the immunoglobulin Fc-5K PEGconjugate at 4° C. for 18 hrs, with the total protein concentration setat 20 mg/ml. For use as a medium for this coupling reaction, a 100 mMpotassium phosphate buffer, pH 6.0, was supplemented with the reducingagent 20 mM SCB. The coupling reaction mixture was purified through twopurification columns. First, a SOURCE Q column (LRC25 85 ml, GEHealthcare) was used to remove the immunoglobulin Fc-5K PEG conjugatewhich remained uncoupled with FVIIa. A salt gradient of 1 M NaCl in 20mM Tris (pH7.5) allowed the immunoglobulin Fc-5K PEG conjugate to beeluted first due to relatively weak binding force, immediately followedby the elution of immunoglobulin Fc-3 arm PEG-FVIIa. Thereafter,secondary purification was conducted to separate immunoglobulin Fc-3 armPEG-FVIIa from FVIIa multimer impurities using a RESOURCE ISO (GEHealthcare) column. The immunoglobulin Fc-3 arm PEG-FVIIa was firsteluted, followed by the FVIIa multimer impurities.

Column: Source Q (LRC25 85 ml, GE Healthcare)

Flow rate: 4 ml/min

Gradient: A 0→7% 1 min B, 7% 37% 80 min B (A: 20 mM Tris pH7.5, B: A+1MNaCl)

Column: RESOURCE ISO (Pre-packed 1 ml, GE Healthcare)

Flow rate: 2 ml/min

Gradient: B 100→0% 60 min A (A: 20 mM Tris pH7.5, B: A+1.6M (NH₄)₂SO₄)

TABLE 4 Active Pharmaceutical Production Yield Ingredient (API) PEG form(API basis) Fac VIIa 2 arm PEG Not prepared 3 arm PEG About above 3%

Example 8: In-Vivo Assay of Immunoglobulin Fc-3 Arm PEG-Octreotide(N)Complex

The immunoglobulinFc-3 arm PEG-Octreotide(N) was assayed forascertaining the effects on body weight and IGF-1 level in SD ratssubcutaneously administered therewith. Octreotide is a potent inhibitorof growth hormone, and is mainly applied to the treatment of acromegaly.It is commercially available from Novartis in two forms: SANDOSTATIN™, ashort acting version; and SANDOSTATIN-LAR™, a long acting version. Now,acromegaly is a syndrome that results upon the overproduction of humangrowth hormone (hGH). In in-vivo tests of octreotide, rats which aresubcutaneously administered therewith are monitored for body weight andIGF-1 level to examine effects thereof on the inhibition of GHsecretion. Likewise, the immunoglobulin Fc-3 arm PEG-Octreotide(N) wasin vivo assayed for changes of body weight and IGF-1 level in rats. Theimmunoglobulin Fc-3 arm PEG-Octreotide(N) was administered in singledoses of 0.5, 1.0, and 2.0 mg/kg while SANDOSTATIN-LAR™ as a control wasadministered in single doses of 1.0 and 2.0 mg/kg, after which the ratswere monitored for body weight and IGF-1 level over two weeks. Both thetwo test materials were subcutaneously administered.

Compared to a vehicle-administered group, the SANDOSTATIN-LAR™ groupswere observed to decrease in body weight by about 3.7% when administeredwith a dosage of 1.0 mg/kg and by about 8.9% when administered with adosage of 2.0 mg/kg. On the other hand, the immunoglobulinFc-3 armPEG-Octreotide(N) groups decreased in body weight by about 44.3% upon0.5 mg/kg administration, by about 40.1% upon 1.0 mg/kg administrationand by about 55.1% upon 2.0 mg/kg administration.

Quantitative analysis for IGF-1 changes in the rats used the rat IGF-1ELISA kit. Compared to the vehicle, the blood IGF-1 level was observedto be decreased by about 15% with SANDOSTATIN 1.0 mg/kg and by about 11%with SANDOSTATIN 2.0 mg/kg on the basis of AUC of blood IGF-1 level. Onthe other hand, the immunoglobulin Fc-3 arm PEG-Octreotide(N) groupsdecreased in blood IGF-1 level by about 23% with a dosage of 0.5 mg/kg,by about 18% with a dosage of 1.0 mg/kg, and by about 25% with a dosageof 2.0 mg/kg [FIG. 2].

The in-vivo test data demonstrates that the immunoglobulin Fc-3 armPEG-Octreotide(N) is more potent in activity than SANDOSTATIN-LAR™, asustained formulation of octreotide.

Example 9: In-Vivo Assay of Immunoglobulin Fc-3 Arm PEG-FSH(N) Complex

In-vivo assay of the immunoglobulin Fc-3 arm PEG-FSH(N) was conductedaccording to the Steelman-Pohley method (Endocrinology 53, 504-616).Immature female SD rats (21 days old) were employed for in vivo assay ofthe immunoglobulinFc-3 arm PEG-FSH(N). The immunoglobulin Fc-3 armPEG-FSH(N) was administered in single doses of 0.018, 0.075, and 0.3μg/rat while Follitrope, a commercially available native form of FSH,was used as a control at dosages of 4, 2 and 1 IU/rat once per day forthree days. A vehicle and the test materials were administered incombination with hCG (human chorionic gonadotropin) at 13.3 U/rat. Eachtest material was subcutaneously administered in an amount of 0.25mL/kg. 72 hours after the first administration of the test materials,the animals were subjected to euthanasia and had an ovariotomy. Theovaries thus excised were weighed.

As depicted in FIG. 3, the immunoglobulin Fc-3 arm PEG-FSH(N) showed invivo activity in a dose-dependent manner, with equality to theimmunoglobulin Fc-2 arm PEG-FSH(N).

What is claimed is:
 1. A protein complex, comprising a physiologically active polypeptide, a dimeric protein, and a non-peptidyl polymer having three functional ends, wherein both the physiologically active polypeptide and the dimeric protein are linked to the respective functional end of the non-peptidyl polymer via respective covalent bond, wherein the dimeric protein is an immunoglobulin Fc domain, and wherein the physiologically active polypeptide is selected from the group consisting of human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon, interferon receptor, colony-stimulating factor, glucagon-like peptide, exendin-4 peptide, ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide), CNP (c-type natriuretic peptide), DNP (dendroaspis natriuretic peptide), G protein-coupled receptor, interleukin, interleukin receptor, enzyme, interleukin-binding protein, cytokine-binding protein, macrophage activating factor, macrophage peptide, B-cell factor, T-cell factor, Protein A, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, transforming growth factor, alpha-1 anti-trypsin, albumin, a-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin, blood factor VII, VIIa, VIII, IX or XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, Protein C, C-reactive protein, renin inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, insulin, somatostatin, octreotide (somatostatin agonist), atriopeptin, cartilage inducing factor, elcatonin, connective tissue activating factor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factors, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin, myostatin, monoclonal antibody, polyclonal antibody, and Fab′ fragment.
 2. The protein complex as defined in claim 1, wherein the immunoglobulin Fc domain is aglycosylated.
 3. The protein complex as defined in claim 1, wherein the immunoglobulin Fc domain is composed of one to four different domains selected from the group consisting of C_(H)1, C_(H)2, C_(H)3 and C_(H)4.
 4. The protein complex as defined in claim 3, wherein the immunoglobulin Fc domain further comprises a hinge region.
 5. The protein complex as defined in claim 1, wherein the immunoglobulin Fc domain is selected from the group consisting of Fc domains of IgG, IgA, IgD, IgE, IgM, and a combination thereof.
 6. The protein complex as defined in claim 5, wherein the immunoglobulin Fc domain is selected from the group consisting of Fc domains of IgG1, IgG2, IgG3, IgG4, and a combination thereof.
 7. The protein complex as defined in claim 5, wherein the immunoglobulin Fc domain is in a dimer or a multimer (combinations of immunoglobulin Fc), each comprising glycosylated immunoglobulin domains from the same origin.
 8. The protein complex as defined in claim 6, wherein the immunoglobulin Fc domain is an IgG4 Fc domain.
 9. The protein complex as defined in claim 8, wherein the immunoglobulin Fc domain is a human aglycosylated IgG4 Fc domain.
 10. The protein complex as defined in claim 1, wherein the non-peptidyl polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, PLA (polylactic acid) and PLGA (polylactic-glycolic acid), lipopolymer, chitin, hyaluronic acid, and a combination thereof.
 11. The protein complex as defined in claim 10, wherein the non-peptidyl polymer is a polyethylene glycol.
 12. The protein complex as defined in claim 1, wherein each of the three functional ends of the non-peptidyl polymer has a functional group selected from the group consisting of an aldehyde group, a propionaldehyde group, a butyl aldehyde group, a maleimide group, and a succinimide derivative.
 13. The protein complex as defined in claim 12, wherein the non-peptidyl polymer has three functional aldehyde groups at the three functional ends.
 14. The protein complex as defined in claim 1, wherein the three functional ends of the non-peptidyl polymer bind to N-terminal, functional groups of the immunoglobulin Fc domain and the physiologically active polypeptide, said N-terminal, functional groups being selected from a group consisting of lysine, histidine, cysteine, and a combination thereof.
 15. The protein complex as defined in claim 1, wherein the physiologically active polypeptide is selected from the group consisting of hormone, cytokine, interleukin, interleukin-binding protein, enzyme, antibody, growth factor, transcription factor, and blood factor.
 16. The protein complex as defined in claim 1, wherein the physiologically active polypeptide is selected from the group consisting of human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon and interferon receptor, colony-stimulating factor, glucagon-like peptide, exendin-4 peptide, ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide), CNP (c-type natriuretic peptide), DNP (dendroaspis natriuretic peptide), G protein-coupled receptor, interleukin and interleukin receptor, enzyme, interleukin-binding protein, cytokine-binding protein, macrophage activating factor, macrophage peptide, B-cell factor, T-cell factor, Protein A, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, transforming growth factor, alpha-1 anti-trypsin, albumin, a-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoeitins, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin, blood factor VII, VIIa, VIII, IX and XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, Protein C, C-reactive protein, renin inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, insulin, somatostatin, octreotide (somatostatin agonist), atriopeptin, cartilage inducing factor, elcatonin, connective tissue activating factor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factors, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin, and myostatin.
 17. The protein complex as defined in claim 16, wherein the physiologically active polypeptide is selected from the group consisting of human growth hormone, interferon-alpha, interferon-beta, granulocyte colony stimulating factor, erythropoietin, exendin-4, imidazole acetyl exendin-4 peptide (exendin-4 agonist), calcitonin, octreotide (somatostatin agonist), and BNP.
 18. A method for preparing a protein complex of claim 1 composed of a physiologically active polypeptide, a dimeric protein and a non-peptidyl polymer having three functional ends, with the linkage of both the physiologically active polypeptide and the dimeric protein to the respective functional end of the non-peptidyl polymer via respective covalent bond, comprising: (1) covalently linking two of the three functional ends of the non-peptidyl polymer to respective N-terminal amino groups of the dimeric protein to form a conjugate, (2) isolating from the reaction mixture of step (1) the conjugate in which the dimeric protein is covalently linked at N-termini thereof with the non-peptidyl polymer and (3) covalently linking the physiologically active polypeptide to one free functional end of the non-peptidyl polymer of the isolated conjugate to obtain the protein complex.
 19. The method as defined in claim 18, wherein the dimeric protein is an immunoglobulin Fc domain.
 20. The method as defined in claim 18, wherein the non-peptidyl polymer has three functional aldehyde groups at the functional ends.
 21. The method as defined in claim 18, wherein the dimeric protein is reacted with the non-peptidyl polymer in step (1) at a molar ratio ranges from 1:2 to 1:5.
 22. The method as defined in claim 18, wherein the conjugate is reacted with the physiologically active polypeptide in step (3) at a molar ratio ranges from 1:0.5 to 1:0.05.
 23. The method as defined in claim 18, wherein the reactions in both steps (1) and (3) are conducted in the presence of a reducing agent.
 24. The method as defined in claim 23, wherein the reducing agent is selected from a group consisting of sodium cyanoborohydride (NaCNBH3), sodium borohydride, dimethylamine borate and pyridine borate.
 25. A composition, comprising the protein complex of claim 1 and optionally a pharmaceutically acceptable vehicle.
 26. The protein complex as defined in claim 1, wherein the protein complex is prepared by the method comprising: (1) covalently linking two of the three functional ends of the non-peptidyl polymer to respective N-terminal amino groups of the dimeric protein to form a conjugate, (2) isolating from the reaction mixture of step (1) the conjugate in which the dimeric protein is covalently linked at N-termini thereof with the non-peptidyl polymer and (3) covalently linking the physiologically active polypeptide to one free functional end of the non-peptidyl polymer of the isolated conjugate to obtain the protein complex. 